In the current application, a collaborative, multidisciplinary research project is proposed that has the potential to create a new paradigm for the study of human physiology in health and disease. A state-of-the-art microfabricated platform will be developed to create a functional, in vitro replica, i.e. simulacrum, of the human colonic epithelium and its associated microbiome. This new technology will be used to perform novel studies and hypothesis testing of intestinal physiology that cannot currently be performed. Furthermore, the technology and protocols developed here will establish the basis for creating organ simulacra from normal and diseased primary human tissues that will change the manner in which studies of tissue function, microbiome influence, and drug effect are performed. Recent progress in organotypic culture of colonic epithelial stem cells has made it possible to create long-lived spheroids in a gelatinous matrix. However, the absence of chemical gradients as well as patterned physical support results in a disorganized and chaotic conformation that poorly mimics the structure and function of the colon, and furthermore is not amenable to microbiome co-culture. We propose to develop a novel microfabricated system that will enable ex vivo culture of human colonic epithelium and overlying microbiota that recapitulates the 3D structure and environment of the colonic mucosa in a setting which lies between an unpolarized cell culture system and the complexity of the intact human organism. Recent technical advances from our labs in sustained monolayer culture of colonic epithelial stem cells will be integrated with microfabricated scaffoldings and devices to create the colonic simulacrum. A number of innovations will be incorporated into the microengineered system with the goal of recapitulating the colonic stem-cell niche, the differentiated intestinal mucosa, the microbiota, and the dynamic information flow between these compartments. The platform will permit tight control of the luminal and basal crypt environments by providing independent fluidic and gaseous access to these compartments. The platform will support formation of mitogen, morphogen, differentiation-factor, dietary-compound and gaseous gradients to enable unprecedented investigations into colonic physiology. A number of hypotheses related to the interplay of dietary factors, the microbiome and the colonic epithelium will be tested to demonstrate the power and broad applicability of this transformative technology.